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. 2021 Oct 31;11(11):1613.
doi: 10.3390/biom11111613.

RadA, a MSCRAMM Adhesin of the Dominant Symbiote Ruminococcus gnavus E1, Binds Human Immunoglobulins and Intestinal Mucins

Marc Maresca  1 , Radia Alatou  2 , Ange Pujol  1 , Cendrine Nicoletti  1 , Josette Perrier  1 , Thierry Giardina  1 , Gwenola Simon  3 , Vincent Méjean  4 , Michel Fons  4
Affiliations

RadA, a MSCRAMM Adhesin of the Dominant Symbiote Ruminococcus gnavus E1, Binds Human Immunoglobulins and Intestinal Mucins

Marc Maresca et al. Biomolecules. .

Abstract

Adhesion to the digestive mucosa is considered a key factor for bacterial persistence within the gut. In this study, we show that Ruminococcus gnavus E1 can express the radA gene, which encodes an adhesin of the MSCRAMMs family, only when it colonizes the gut. The RadA N-terminal region contains an all-β bacterial Ig-like domain known to interact with collagens. We observed that it preferentially binds human immunoglobulins (IgA and IgG) and intestinal mucins. Using deglycosylated substrates, we also showed that the RadA N-terminal region recognizes two different types of motifs, the protein backbone of human IgG and the glycan structure of mucins. Finally, competition assays with lectins and free monosaccharides identified Galactose and N-Acetyl-Galactosamine motifs as specific targets for the binding of RadA to mucins and the surface of human epithelial cells.

Keywords: Caco-2; HT-29-16E; Ruminococcus gnavus; adhesin; bacterial Ig-like domain; collagen; mucin; mucus; solid phase assay.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of the radA cluster (A) and of the RadA domains (B). In Figure 1A, radA, orfX and srtB coding sequences are represented as black arrows and intergenic regions as grey lines. radA, orfX and srtB length (5310 bp, 630 bp and 669 bp, respectively) is not scaled. ↱ stands for putative promotors. ∏ indicates putative Rho-independent transcription terminators and the corresponding theoretical ΔG0 expressed as kCal/mol. In Figure 1B, □ represents the CnaB domains, ↓ the LPQTP cell wall anchor domain, and ▼ the C-terminal transmembrane domain.
Figure 2
Figure 2
Schematic localization of the PCR-primers along the radA gene (A) and expression of radA (B). In situ: RT-PCR on total RNA extracted from E1-mono-contaminated rats. In culture: RT-PCR on total RNA extracted from E1 culture in BHI-YH medium. Control: PCR on E1 chromosome DNA. Lines 1, 2, 3 and 4 correspond to PCR reactions carried out with couples of primers Ad5F-Ad6R, Ad3F-Ad4R, Ad2F-Ad3R and Ad1F-Ad2R, respectively. M stands for the GeneRuler 1kb DNA Ladder marker. 1000 bp and 800 bp with arrows point to the size of the products.
Figure 3
Figure 3
Analysis of purified GST-RadA35-252, by SDS-PAGE. SM, standard marker; Rad, GST-RadA35-252; GST, glutathione S-transferase; 25 kDa, 50 kDa and 75 kDa indicate the molecular weight of three proteins present in the SM.
Figure 4
Figure 4
GST-RadA35-252 binds preferentially to human immunoglobulins and mucins. The interaction of GST-RadA35-252 with collagen I, collagen IV or gelatin (A) immunoglobins from various animal species (B) or with mucins type I (MUC1), type II (MUC2) or type III (MUC3) (C) was studied by the solid-phase binding assay as described in Material and Methods. All tested proteins were coated at 4 pmol per wells (40 nM). Results are expressed as means ± S.D. (n = 3).
Figure 5
Figure 5
The interaction of GST-RadA35-252 with MUC2 depends on a glucidic motif. The binding of WGA (A) or GST-RadA35-252 (B) to native and deglycosylated (DG) collagen IV, human IgG or mucin type II (MUC2) (coated at 4 pmol per wells (40 nM)) was measured as explained in Materials and Methods. Results are expressed as means ± S.D. with *: p < 0.05; **: p < 0.01 (n = 3).
Figure 6
Figure 6
The interaction of GST-RadA35-252 with collagen IV, human IgG or type II mucin involves two separate domains/mechanisms. GST-RadA35-252 was pre-incubated with soluble collagen IV, human IgG or type II mucin (MUC2) (400 nM) before being added to wells coated with collagen IV, human IgG or type II mucin (MUC2) (protein-coated at 4 pmol per wells (40 nM)) as explained in Materials and Methods. Results are expressed as means ± S.D. with *: p < 0.01, **: p < 0.001 (n = 3).
Figure 7
Figure 7
Galactose and N-acetyl-Galactosamine in solution inhibit the interaction of GST-RadA35-252 with type II mucin. GST-RadA35-252 was incubated with monosaccharides in solution before being added to coated type II mucin (MUC2) (coated at 4 pmol per wells (40 nM)) as explained in Materials and Methods. Tested monosaccharides were Fructose (Frc), Fucose (Fuc), Mannose (Man), Sialic acid, Galactose (Gal), N-acetyl-galactosamine (GalNAc), Glucose (Glc) and N-acetyl-glucosamine (GlcNAc). Results are expressed as means ± S.D. (n = 3).
Figure 8
Figure 8
Lectins binding Galactose and N-acetyl-Galactosamine inhibit the interaction of GST-RadA35-252 with type II mucin. Coated type II mucin (coated at 4 pmol per wells (40 nM)) was pre-incubated with various lectins: Wheat Germ Agglutinin (WGA, specific for GlcNAc), Ulex Europaeus Agglutinin (UEA, specific of Gal) and Jacalin (JAC, specific of Gal and GalNAc) (all at 100 µg/mL). A solid-phase binding assay of GST-RadA35-252 was then conducted as explained in Materials and Methods. Results are expressed as means ± S.D. *: p < 0.01 (n = 3).
Figure 9
Figure 9
GST-RadA35-252 binds to the surface of human intestinal epithelial cells. Caco-2 and HT29-16E cells were used to study the dose-dependent binding of GST-RadA35-252 to human intestinal cells (A). The inhibitory effect of lectins and monosaccharides on the binding of RadA35-252 to Caco-2 cells (B) or HT29-16E cells (C) was studied as indicated in the Materials and Methods. Results are expressed as means ± S.D. *: p < 0.05, **: p < 0.01 (n = 3).
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